Insulin highly respirable microparticles

ABSTRACT

The invention describes novel dried powders of peptide therapeutic agent useful for producing highly respirable aerosols and the methods for their manufacture. Insulin is the peptide therapeutic agent in the preferred embodiment. The powders of insulin prepared for pulmonary administration are characterized by the peculiar structure and shape of the microparticles that allow the powder to flow and to be easy aerosolized. Typical dry powder of insulin described in this patent show corrugated, nonagglomerated microparticles with a low tapped density. The mean geometric diameter (particle size) ranges between 1.0 and 10.0 micron and the mass median aerodynamic diameter (MMAD) ranges between 1.0 and 4.0 micron. These insulin pulmonary powders exhibit in vitro a very high respirable fraction (&gt;75%).

FIELD OF THE INVENTION

It is known that certain drugs delivered to the lung are readilyabsorbed through the alveolar region into the blood circulation.Pulmonary delivery is a promising alternative route in particular forthe administration of macromolecules such as proteins, polypeptides andnucleic acids, which are exclusively delivered by injection. Lungdelivery is useful for both systemic and local therapeutic activity.Pulmonary drug delivery has to be achieved by producing an aerosol ofthe active. Aerosols can be generated by different methods, includingliquid nebulizers, pressurized metered dose inhalers (MDI) and drypowders inhalers (DPI). The CFC propellant phase out caused aerosolbased MDI to loose favor while increasing interest focused on dry powderdevices. In such devices, drugs are formulated as respirable dry powderobtained by freeze-drying or spray-drying or other suitable techniques.The drugs may be combined with safe excipients in order to improve theirrespirability, stability and flowability.

Typical techniques for delivering dry powder formulations via a DPI areloading one dose of the drug in a hard gelatin capsule or aluminumblister or to load the device with multiple doses leaving to the deviceto sample the amount required. This step requires powders havingfavorable properties of flow and packing. These properties are typicalof the coarse powders. Upon patient inspiration the air flowing throughthe device penetrates into the drug reservoir and aerosolizes the powderdose. This second step requires micronized powders having particle withsize, shape and density useful for aerosolization.

The ability to deliver a drug to the alveolar region of the respiratorytract, where the absorption takes place, is problematic under differentpoints of view. In details, a powder must be capable to overcome theparadox to be fine for aerosolization and lung deposition but at thesame time to be coarse for the dosing in the device of the amount to beadministered. Firstly, the dose of drug must be accurately metered andthis relies on the packing and flow properties of the powder andcharacteristics of device for administration. Secondly, the powder to beinhaled must be easily dispersed in order to assure the generation ofrespirable aerosol and this relies on powder size, shape and density. Asolution for these two contradictory aspects is the object of thispatent.

DESCRIPTION OF RELEVANT LITERATURE

The delivery of insulin to the lung has been proposed since itsdiscovery. The simplest method to deliver insulin to the lung inpreclinical studies was by direct intra-tracheal instillation of anaqueous formulation. In this case, distribution in the lung tends to belocalized and uneven than that seen after aerosol administration,resulting in small surface area available for the absorption. A. L.Jones (Proceedings of the third European congress of Biopharmaceuticsand Pharmacokinetics, Vol 2, page 143-149) in 1987 reported a rapidabsorption of insulin in rats. P. Colthorpe and S. Farr in 1992(Pharmaceutical Research, 9: 764-768) using pharmaco-scintigraphycompared the deposition and absorption of the direct instillation andthe nebulization of an acidic insulin solution. He elegantlydemonstrated that the absorbed fraction for aerosolized insulin was 10fold greater than instilled insulin. This provides a clear proof how themode of administration profoundly affects the fate of pulmonarydelivery.

Using intratracheal instillation, the effect of formulation relatedvariables on the extent of pulmonary insulin absorption has also beeninvestigated, including osmolarity, viscosity and solution pH. Accordingto F. Komoda (J. Pharm. Sci. 1994, 83, 863-867) insulin formulation atpH 3 showed greater bioavalability than those at pH 7 afterintratracheal instillation. They explained the result by the inhibitionof insulin association in the lower pH formulation. Insulin exists asmonomer, dimer and exhamer. Dimers and exhamers come from aggregation ofthe monomeric form and the relative percentage in solution of threeforms depends on pH and concentration of the hormone. It is known thatinsulin self-aggregation affects its diffusive transport acrossbiological membrane (Diabetes Care 1990,13,953-954).

Many ways have been suggested for aerosolizing insulin in form ofsolution, dry powders and even suspension of liposomes. Metered doseinhalers and Dry powder inhalers are the most recent devices forpulmonary administration of drugs. Metered dose inhalers for deliveringcrystalline insulin suspended in a propellant have been proposed by S.Lee (J. Pharm. Sci. 1976: 65, 567-572) and a patent exists on this field(U.S. Pat. No. 5,320,094). Dry powders inhalers carrying insulin arealso described in the literature (for a complete review: J. S. Patton:Inhaled Insulin, Adv. Drug Del. Rev. 35, 1999, 235-247). Pulmonarydelivery of dry powder medicaments in large particle porous particleshas been investigated by R. Langer and co-workers (J. App. Physiol.1998: 85, 379-385), and patented (WO9966903). Others preparations forinhalation which comprise insulin and a substance which enhances theabsorption of insulin in the lower respiratory tract, have been proposedin the form of a powder preparations suitable for inhalation (U.S. Pat.No. 6,306,440). Intranasal and respiratory delivery of a variety ofpolypeptides, including insulin, in the presence of an enhancer has beenalso described by T. Nagai (J. Contr. Rel. 1984:1, 15-22) and L. Rydén(Int. J. Pharm. 1982: 83, 1-10) and in several patents releasedworldwide (WO9302712, WO9102545, WO 9009780, WO8804556).

The preparation of an amorphous powder containing insulin wasillustrated in a patent which described the methods for spray dryingpolypeptide, polynucleotide and other liable drugs together with acarrier to improve stability of the active after drying (EP 0520748).The first patent on insulin medical aerosol formulation (EP 0655237)discloses the production of an aerosol containing also spray driedinsulin intended for lung administration. In the example 4 of the citedpatent a powder is prepared from an alcoholic (25% v/v) solutioncontaining the same amount of insulin and lactose and 0.1% soya beanlecithin. In another patent (WO A 9524183) insulin is produced in formof a dried powder from buffered solution at pH 6.7±0.3 containing thehormone. This patent discloses the use of a citrate buffer to dissolvecrystal insulin and subsequently the powder is produced by spray drying.Some criticism (refers to WO00/00176, page 2, line 4-line 6) has beenaddressed to the experimental section.

Finally, dry powders inhalers are disclosed in several patentapplications. Manual pumps (U.S. Pat. No. 3,921,637) or multiplereceptacle disks or strips (EP 0467172) are employed. Puncturing gelatincapsule disperser is described somewhere else (U.S. Pat. No.4,338,9314). A held-pump device has been also patented (WO09007351).Independently on the device used, the characteristics of drug in powderform are crucial for the efficacy of the preparation.

OBJECT OF THE INVENTION

The object of present invention are pharmaceutical powders of insulinsuitable for pulmonary administration intended for the long-termtreatment of diabetes, characterized by a structure of themicroparticles composing the powder that imparts an elevatedrespirability, together with favorable flow and packing characteristics.The powders obtained through have been produced by spray drying in neverexplored conditions of manufacturing, in particular at pH lower than theisoelectric point of insulin. The acidic pH used for particlepreparation would allow to obtain higher absorption as F. Komodaprevioulsy demonstrated.

The pulmonary powders of insulin object of this patent are characterizedby structure and shape of the constituent microparticles definedcorrugated or raisin like, completely different from the insulinmicrocrystal structure. These pulmonary powders of insulin show a flowand packing characteristics that allow them to be directly introduced inthe reservoir of DPI delivery systems. More relevant is the fact thatthe respirability of the powders having these properties is higher thatthe usual values described in literature. In fact, the fraction ofrespirable particles composing the insulin aerosol produced with thepowders here described ranged between 83.9% and 90.4%, whereas commonvalues range between 20-40%.

This respirability has been assessed using the Andersen Cascade Impactoras described in the European Pharmacopeia (4th Edition, <2.9.18>page216). This apparatus is used to determine the fine particles of anaerosol cloud, generated by preparations for inhalation, and allows themeasure of the mass of drug less than a particular aerodynamic particlesize. The mass of drug having aerodynamic diameter lower than 5 μm isgenerally considered as “respirable”, even though the optimal size foralveolar deposition is in the range 5-2 micron.

The manufacturing procedures described in this patent provide theproduction of fine powders in which more than 90% of particles havedimensions less than 9 microns as volume diameter. The method forpreparing said formulation allows high percentage of pharmaceuticalactivity of the powder to be manufactured.

Moreover, the dry powder exhibits adequate chemical and physicalstability. In the background art the insulin solutions to be spray-driedare reported to have a pH near the neutrality obtained by using citratebuffer (pH 6.7±0.3, WO-A-95/24183) or above neutral (WO00/00176).Insulin for pulmonary therapy in patent WO-A-95/24183 was declared asprepared by spray drying solutions in physiologically acceptable buffersuch as citrate buffer at pH between 2.0-9.0, but the powder describedin the example was made by spray drying a solution at pH 6.7±0.3. Inpatent WO00/00176 the spray drying of a true solution over theisoelectric point of insulin was described.

The present invention is based on the surprising discovery that thespray drying of clear, concentrated, aqueous solutions of insulin havingpH lower than the isoelectric point of the hormone (5.4), produces veryhigh respirable dry powders. These powders can be obtained fromun-buffered or volatile buffer solutions and were never prepared before.The spray drying of insulin solution under the isoelectric point andtherefore, in acidic conditions and without the use of permanentbuffering agents, was not previously considered likely for stability andrespirability reasons. On the contrary, insulin microparticles producedby spray drying acidic solutions of the hormone resulted in powdersparticularly suitable for lung administration, because they exhibit ahigh respirable dose. In addition, the stability was acceptable inrefrigerated conditions but, when the powder was manufactured from asolution in acetic acid (volatile buffer), the stability resultedsurprisingly very high also in normal conditions. Since no permanentbuffers were used, it is also expected that these powders inhaled at thetherapeutic doses do no modify the alveolar surfactant pH.

As described in previous patent (WO00/00176) concerning insulinformulations obtained by spray drying, the neutralisation procedure ofinsulin solutions to pH above 7.0 results in the production ofmicroparticles with “dimpled surface that may be beneficial” in term ofrespirability. We reproduced these particles made at pH 7.45 in order tofocus the “dimple” shape. The procedure applied is the one described inthe WO00/00176 patent. The microparticles obtained according to thepresent patent are much more than dimpled since they are corrugated orraisin like (FIGS. 1 and 2). In addition, they are non-cohesive withfavourable aerodynamic size and density characteristics.

Similar shapes were observed independently of the acids employed for thepreparation of insulin solutions. However, we unexpectedly discoveredthat when the volatile organic acetic acid is used to dissolve insulin,powders obtained by spray drying from these low pH solutions have lostacidity. In fact, the dissolution of these powders in distilled degassedwater gives rise to a solution having pH higher than the value of theoriginal solution. This fact made this powder chemically very stableduring storage.

Therefore, the novelty of the powders described in this patent is basedon the finding that the peculiar corrugated microparticles obtained byspray-drying insulin acidic solution are micronized, free flowing andwith low tapped density. The microparticles are essentially amorphousand characterized by a shape defined corrugated or raisin like. Thisparticle shape makes the powders not cohesive since the microparticlesmaintain their individuality and do not agglomerate. In addition, theyshow substantially no losses of activity if stored in refrigeratedconditions, but when they are prepared from acetic acid, the insulinpowders are very stable also at room conditions (25° C.). In particular,we surprisingly discover that the acetic acid solution of insulin at pH3.3 after spray drying gave rise to a powder that re-dissolved indistilled degassed water at 1 mg/ml showed a pH of 6.4. Surprising thispowder shows a superior stability at 25° C. allowing the preparation tobe used and dispensed at room conditions, in comparison with insulinspray dried powders prepared with HCl that must be stored atrefrigerated conditions.

Differently from the common technique, such spray dried powderscharacterized a peculiar insulin particle shape, are produced fromclear, volatile buffer or un-buffered solutions having acidic pH valuesbetween 3.0-4.5, lower than the isolelectric point of insulin. The useof acidic solutions avoids the risk of precipitation induced byincreasing the pH above insulin isoelectric point up to neutrality, butmore interestingly provides a structure to dried product thatsurprisingly is very useful for the aerosolization. In fact, thesepowders other than to be micronized, are not cohesive, quite freeflowing and easy meterable in the DPI. These physical properties,together with the favourable aerodynamic behaviour due to the size,shape and density of the particles, determine an unexpected andsurprisingly high respirability.

Finally, the powders contain residual moisture enough to preventexcessive degradation and they can be stored at normal humidity andtemperature conditions when prepared from volatile acetic acid.

FORMULATION ASSAY

The activity of all formulations has been estimated by HPLC. Accordingto the official monographs for “Insulin preparations” (USP 26 andEuropean Pharmacopoeia 4th Edition, page. 1368-1381) HPLC performed indifferent conditions gives information about potency (according USP 26,potency is evaluated in comparison to a certified standard, testdescribed under “ASSAY”), purity (quantification of the “relatedproteins”) and about the presence of covalent aggregation (called“impurities with molecular masses greater than that of insulin”), bothin European Pharmacopoeia 4th Edition.

Pharmacopoeias limits and specifications for the insulin preparationsare: A21 desamido not more than 5% of total area of peaks, other notmore than 6%. Impurities with molecular masses greater than that ofinsulin: not more than 2% of total area of peaks.

Aerodynamic diameter was assessed using the Andersen Cascade Impactor.The percentage of mass less than the stated aerodynamic diameter versusaerodynamic diameter is then plotted on a log probability paper (USP 26page 2123). Respirability of the described formulations is derived fromthe data plotted as described above considering the mass less than 5microns as respirable. Packing properties have been studied using tapdensity measurements. According to the official monograph, tap densitieshas been evaluated after 1250 taps (USP 26) employing a 10±0.05 mlcylinder filled with the powder.

DESCRIPTION OF THE INVENTION

The microparticles of this invention contain drug substantially free ofexcipients, but in certain conditions mixture of drug and diluent suchas mannitol can be used. Substantially free of excipients means that themicroparticles of the invention can include process-linked component ashydrochloric acid or acetic acid and eventually their sodium salts incase of pH adjustement up to about 10% of the total solids. The mainadvantage of the use of substantially excipient-free formulations isthat each dose can contain a large amount of the active. Buffer saltslike citrate are not necessary both for solubility and stability of themicroparticles described under the invention. In all previous patentsinsulin final solutions for spray drying are reported to have a pH nearthe neutrality using citrate buffer (6.7±0.3, WO-A-95/24183) or aboveneutral (WO00/00176). The procedure at pH 6.7 did not allow themicroparticles to be produced from a clear solution of insulin and fromthe clear solution above the neutrality the particles had a surfacesmooth or “dimpled”, morphological characteristics, considered asbeneficial for inhalation. Nevertheless, the use of mild acidicsolutions leads to reproducible production of corrugated or raisin likemicroparticles, without affecting the insulin stability if properlystored.

The solutions of insulin are spray dried in a conventional spray dryingapparatus; even rotary atomization, pressure atomization and two-fluidatomization can be employed as spraying process. No particularrestrictions are placed on the gas used to dry the sprayed materials.Filtered air is used in the manufacturing methods described below. Thetemperature of the inlet of the gas used to dry the sprayed materialsshould be chosen so that it does not cause degradation of the active.The range may vary between 50° C. and 200° C. The temperature of theoutlet gas used may vary between 30° and 100° C., preferably in therange 40° C. and 60° C. This has been found to affect more than theinlet temperature set up the degree of degradation of the dried product.The fact that inlet and outlet temperature above 50° C. can be used hasbeen noted and reported (U.S. Pat. No. 6,582,728). Insulin solutionswere prepared by dissolving from 5 to 20 mg of the hormone permilliliter in 10⁻² M hydrochloric acid solutions or 0.4M diluted aceticacid solutions (pH 2.6), diluting with distilled water and adding ifnecessary an adequate amount of NaOH solution to pH between 3.0 and 4.5,in any case avoiding insulin precipitation. Excipients such aspolyalcohols can be added before pH correction. A Mini Spray DrierBuchi, model 191 (Buchi, Labortechnik A G, Flawil, Switzerland)equipment was used. The inlet air (drying gas) had initially, beforeheating, a relative humidity of about 30-70%. The nozzle was providedwith an orifice of 0.7 or 1.0 mm internal diameter. The atomizing gaswas filtered compressed air. The spray drier was equipped with astandard cyclone. The used range of the spray drying parameters was:feed flow rate 180-360 ml/h; nozzle gas flow rate 500-800 I/h; inlet airtemperature <140° C., producing an outlet temperature of 40-60° C.;aspirator capacity <35 m³/h (100% setting). The selected parameter rangeallowed to obtain an original and new powder with good flow and packingproperties, having particle size, shape and density in the respiratoryrange and with a respirable fraction higher than 80%, with a moisturecontent in the range of 2 to 8% and a pH of reconstituted solution nearneutrality when the acidic solution to spray dry was made with aceticacid, thus preventing insulin degradation.

EXAMPLES

According to the object of the invention, the following dispersible drypowder formulations were prepared as described. All formulations meetthe strict specifications for content and purity required forpharmaceutical products.

Example 1

A) Formulation.

2550 mg of highly purified bovine insulin was dissolved in 200 ml ofaqueous 10⁻²M hydrochloric acid. The solution was added with 100 ml ofdistilled water and then with 450 mg of mannitol under stirring to givea final solid concentration of 10 mg per ml (8.5 mg bovine insulin perml). The pH of the clear solution was adjusted to 4.35 using NaOH 0.1 Ndropwise.

B) Spray Drying.

This solution was filtered and was spray dried using a Mini spray drierBüchi, model 191 (Büchi, Labortechnik A G, Flawil, Switzerland) underthe following process conditions: feed flow rate 195 ml/h; nozzle gasflow rate 600 nl/h; atomizing nozzle diameter 1.0 mm; inlet airtemperature 120° C., producing an outlet temperature of 42° C.;aspirator capacity 100% setting. The yield was about 60%.

C) Characterization.

The collected powders were assayed by HPLC for covalent aggregation anddegradation products (A21 desamido insulin) according to the EuropeanPharmacopeia 4 (pag. 1368-1381), by Scanning Electron Microscopy (SEM)for morphology investigations, by laser diffraction for particle sizedistribution and Andersen Cascade Impactor for respirable fractionevaluation.

The potency was 22.6 Ul/mg, the related proteins were 0.5% and theimpurities with molecular mass greater than insulin were 0.55%. Thepowder contained approximately 5.8% moisture. The particle sizedistribution of the powder was determined to be 2.33 (d₁₀), 3.62 (d₅₀)and 5.68 (d₉₀) microns as volume diameter. The respirability (mass lessthan 5 microns) derived from Andersen Cascade Impactor data was high as85.7%. The packing properties measured as tapped density (European.Pharmacopoeia 4 t Edition) was 0.2 g/cm³. The powder dissolved indistilled and degassed water gave rise to a pH of 4.4. Weighed amountsof powder were then placed into separated glass vials and stored at −18°C.,in a refrigerator at 5±3° C. and at room temperature and humidity(25±3° C. and 65±5% RH respectively) and analysed at different times byHPLC for stability.

Example 2

A): Formulation.

975 mg of bovine insulin was dissolved in 95 ml of aqueous dilutedacetic acid (pH 2.6). The solution was added with 0.6 ml of NaOH 1 M.The pH of the clear solution was 3.27.

B): Spray drying Process.

This solution was filtered and subsequently spray dried using a Minispray drier Büchi, model 191 (Büchi, Labortechnik AG, Flawil,Switzerland) under the following process conditions: feed flow rate 200ml/h; nozzle gas flow rate 500 nl/h; atomizing nozzle diameter 1.0 mm;inlet air temperature 130° C., producing an outlet temperature of 55°C.; aspirator capacity 100% setting. The yield was about 50%.

C) Characterization.

The collected powders were assayed by HPLC for covalent aggregation anddegradation products (A21 desamido insulin) again according to theEuropean Pharmacopeia and by the already quoted assays. The potency was28.9 Ul/mg, the related proteins were 0.6% and the impurities withmolecular mass greater than insulin was 0.33%. The particle sizedistribution was determined to be 4.06 (d₁₀), 4.36 (d₅₀) and 4.93 (d₉₀)microns as volume diameter. The respirability (mass less than 5 microns)derived from Andersen Cascade Impactor data was high as 83.9%. Thetapped density, index of packing properties, (Eu.Pham), was 0.1 g/cm³.The powder dissolved in distilled and de-gased water gave rise to a pHof 6.4. Microparticles exhibit a raisin-like shape (FIG. 1).

Example 3

A) Formulation. 1750 mg of highly purified bovine insulin was dissolvedin 120 ml of aqueous 10⁻² M hydrochloric acid. The solution was addeddropwise with0.0.7 ml of 1 N NaOH to give a final pH of about 4.44. Thesolution is clear and contains about 15 mg per ml of solids.

B) Spray Drying Process.

This solution was filtered and then spray dried using a Mini spray drierBüchi under the following process conditions: feed flow rate 195 ml/h;nozzle gas flow rate 600 nl/h; atomisng nozzle diameter 1.0 mm; inletair temperature 120° C., producing an outlet temperature of 46° C.;aspirator capacity 100% setting. The yield was about 55%.

C) Characterization.

The microparticles were assayed by HPLC for covalent aggregation anddegradation products (A21 desamido insulin) according to the EuropeanPharmacopoeia, by SEM for morphology investigations, by laserdiffraction for particle size distribution and by Andersen CascadeImpactor for respirable fraction evaluation.

The potency was 27.1 Ul/mg, the related proteins were 0.7% and theimpurities with molecular mass greater than insulin were 0.4%. Theformulation contained approximately 4.7% moisture

The particle size distribution was determined to be 3.12 (d₁₀), 4.72(d₅₀) and 7.24 (d₉₀) microns as volume diameter. The powder dissolved indistilled and degassed water gave rise to a pH of 4.7.

The respirability (mass less than 5 microns) derived from AndersenCascade Impactor data was high as 90.4%.

Weighed amounts of powder were then placed into separated vials andstored at −18° C., in a refrigerator at 5±3° C. and at room temperatureand humidity (25±3° C. and 65±5% RH respectively) and analyzed atdifferent times by HPLC for stability.

1-16. (canceled)
 17. Microparticles stable at room temperature ofinsulin, optionally in association with excipients selected from thegroup consisting of saccharides, polysaccharides, aminoacids,phospolipids and polyalcohol, said microparticles: being obtained byspray drying an aqueous solution of insulin having an acid pH under theisoelectric point (5.4) of insulin and a concentration of insulin inamounts of from 5 to 100 mg/ml, showing a d90 volume diameter lower than9 μm, 80% of them exhibiting an aerodynamic diameter lower than 5 μm,containing less than 10% by weight of salts, wherein said aqueoussolution of insulin to be spray dried is prepared in an unbufferedaqueous solution of acetic acid.
 18. Microparticles according to claim17 having a tapped density lower than 0.2 g/cm3.
 19. Microparticlesaccording to claim 18, wherein said excipient is mannitol. 20.Microparticles according to claim 17 containing insulin in amorphousform.
 21. Pharmaceutical compositions suitable to be inhaled containingthe microparticles according to claim
 17. 22. Pharmaceuticalcompositions suitable to be inhaled consisting of the microparticlesaccording to claim 17.